CN111107902A

CN111107902A - Small molecule sensitization to BAX activation for induction of cell death

Info

Publication number: CN111107902A
Application number: CN201880031879.2A
Authority: CN
Inventors: L·D·瓦伦斯基; J·普里茨; F·沃切特
Original assignee: Dana Farber Cancer Institute Inc
Current assignee: Dana Farber Cancer Institute Inc
Priority date: 2017-03-14
Filing date: 2018-03-14
Publication date: 2020-05-05
Also published as: JP2020514367A; CA3054452A1; EP3595777A1; AU2018236233A1; EP3595777A4; WO2018170067A1; US20200172530A1

Abstract

Provided herein are compositions comprising a compound of formula I, II or III and compositions comprising a compound of formula IV useful for sensitizing and/or activating the pro-apoptotic activity of BAX. Also provided are methods of treating a BAX-associated disease (e.g., cancer) and methods of identifying compounds that sensitize and/or activate the pro-apoptotic activity of BAX polypeptides.

Description

Small molecule sensitization to BAX activation for induction of cell death

Federally sponsored research or development

The invention was made with government support under grant No. NIH1R35, CA197583 awarded by the national institutes of health. The government has certain rights in the invention.

Technical Field

Provided herein are compositions containing a compound of formula I, formula II, or formula III, as well as compositions containing a compound including a moiety of formula IV, which are useful for sensitizing and/or activating the pro-apoptotic activity of BAX. Also described are methods of treating a BAX-associated disease (e.g., cancer) and methods of identifying compounds that sensitize and/or activate the pro-apoptotic activity of BAX polypeptides.

Background

BAX is a 21kDa globular protein composed of 9 α -helices and functions as a key effector of the BCL-2 family regulated mitochondrial apoptotic pathway the α 5/α 6 hairpin forms the hydrophobic core of the protein, the juxtaposition of α -helix 1 and α -helix 6 creates a ligand-interacting surface that regulates the initiation of BAX activation, and on the opposite side of the protein, the self-inhibited α 9 helix is present in the hydrophobic groove composed of portions of α -helix 2, α -helix 3 and α -helix 4 (see, e.g., Suzuki et al, Cell,200,103: 645-.

Disclosure of Invention

The present application provides, inter alia, compositions comprising: a compound of formula I or a pharmaceutically acceptable salt thereof,

and one or more pharmaceutically acceptable carriers, wherein: l is¹Selected from the group consisting of bond, C_1-3Alkylene, -O-, -O (C)_1-3Alkylene) -, C_1-3Cyanoalkylene, -S-, -SO₂-、-S(C_1-3Alkylene) -and-c (o) -or a pharmaceutically acceptable salt thereof; r¹Selected from halogen, OH, C_1-3Alkyl radical, C_1-3Haloalkyl, NH₂CN, phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein said phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl are each optionally substituted with 1,2 or 3 independently selected R^ASubstituted by groups; r²Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; r³Selected from H, halogen, OH, NH₂、C(O)C_1-3Alkyl and C (S) C_1-3Alkyl groups; r⁴Selected from H, halogen, OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Haloalkyl and O (C)_1-3Cyanoalkyl); r⁵Selected from H, halogen, OH, NH₂And C (O) C_1-3Alkyl groups; r⁶Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; each R^AIndependently selected from OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Hydroxyalkyl, C (O) OH, C (O) C_1-3Alkyl and C (O) N (C)_1-3Alkyl radical)₂Wherein said C_1-3Alkyl is optionally substituted by NH₂And (4) substitution.

In some embodiments, L of formula I¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-C (O) -in a pharmaceutically acceptable carrier. In some embodiments, L is¹is-O-, -CH₂-or-OCH₂-。

In some embodiments, R of formula I¹Selected from the group consisting of Cl, CH₃、CF₃、NH₂CN, phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein said phenyl, 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl are each optionally substituted with 1 or 2 independently selected R^AAnd (4) substituting the group.

In some embodiments, R of formula I¹Selected from the group consisting of Cl, CH₃、CF₃、NH₂CN, phenyl, pyridyl, furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, pyrazolyl, 1,2, 4-thiadiazolyl, piperidinyl, morpholinyl, and 4, 5-dihydrothiazolyl, wherein said phenyl, pyridyl, furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, pyrazolyl, 1,2, 4-thiadiazolyl, piperidinyl, morpholinyl, and 4, 5-dihydrothiazolyl are each optionally substituted with 1 or 2 independently selected R^AAnd (4) substituting the group.

In some embodiments, each R of formula I^AIndependently selected from OH, NH₂、CN、CH₃、CH₂OH、CH₂CH₂NH₂、C(O)OH、C(O)CH₃And C (O) N (CH)₃)₂Group (d) of (a).

In some embodiments, R of formula I¹Is phenyl, optionally substituted by 1 or 2 independently selected R^AAnd (4) substituting the group. In some embodiments, R of formula I¹Is phenyl, 4-hydroxyphenyl, 3-hydroxyphenyl, 4-aminophenyl, 4-carboxyphenyl or 4-hydroxymethylphenyl.

In some embodiments, R of formula I²Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a). In some embodiments, R of formula I²Is H or CH₃。

In some embodiments, R of formula I³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃Group (d) of (a). In some embodiments, R of formula I³Is H.

In some embodiments, R of formula I⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN. In some embodiments, R of formula I⁴Is H or OH.

In some embodiments, R of formula I⁵Selected from H, F, Cl, NH₂And C (O) CH₃Group (d) of (a). In some embodiments, R of formula I⁵Is H or NH₂。

In some embodimentsIn the formula I, R⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a). In some embodiments, R of formula I⁶Is H.

In some embodiments, the compound of formula I is selected from the group consisting of:

in some embodiments, the compound of formula I is:

or a pharmaceutically acceptable salt thereof.

The present application also provides a composition comprising: a compound of formula II or a pharmaceutically acceptable salt thereof,

and one or more pharmaceutically acceptable carriers, wherein: x¹Is NH or S; x²Is C or N; l is¹Selected from the group consisting of a bond, -C (O) -, -C (O) O-and-SO₂-a group of compositions; r¹Selected from the group consisting of C_1-3Alkyl, NH₂Two (C)_1-3Alkyl) amino and 5-6 membered heterocycloalkyl; r²Selected from H, halogen, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; r³Selected from the group consisting of H, C_1-3Alkyl and 5-6 membered heteroaryl; when X is present²When is N, R³Is absent; r⁴Selected from H and C_1-3Alkyl groups.

In some embodiments, X of formula II¹Is NH.

In some embodimentsIn (II) X¹Is S.

In some embodiments, X of formula II²Is C.

In some embodiments, X of formula II²Is N.

In some embodiments, R of formula II¹Is selected from the group consisting of CH₃、CH₂CH₃、NH₂、N(CH₂CH₃)₂Piperidinyl and dihydrothien-3 (2H) -keto (onyl).

In some embodiments, a-L of formula II¹-R¹Form a radical selected from the group consisting of NH₂、C(O)OCH₃、C(O)OCH₂CH₃、C(O)N(CH₂CH₃)₂、SO₂-piperidinyl and dihydrothiophen-3 (2H) -onyl groups.

In some embodiments, R of formula II²Selected from H, Cl, CH₃And C (O) OCH₂CH₃Group (d) of (a).

In some embodiments, R of formula II³Selected from the group consisting of H, CH₃、CH₂CH₃And thienyl.

In some embodiments, R of formula II⁴Selected from H and C_1-3Alkyl groups.

In some embodiments, the compound of formula II is selected from the group consisting of:

the present application also provides a composition comprising: a compound of formula III or a pharmaceutically acceptable salt thereof,

and one or more pharmaceutically acceptable carriers, wherein:

a single bond or a double bond; ring A is selected from the group consisting of 5-6 membered cycloalkyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, forming a fused ring with ring B, wherein ring A is optionally substituted with 1,2 or 3 independently selected R^ASubstituted by groups; r¹Selected from the group consisting of H, C (O) OC_1-3Alkyl, OC (O) C_1-3Alkyl, C (S) NH₂And ═ N-OH; r^1aIs H; when R is^1aWhen the carbon atom to which it is attached forms a double bond, R^1aIs absent; r²Selected from the group consisting of H and halogen; r^2aIs H; when R is^2aWhen the carbon atom to which it is attached forms a double bond, R^2aIs absent; r³Selected from H, halogen, C_1-3Alkyl radical, C_1-3Hydroxyalkyl, NHC (O) C_1-3Alkyl and (C)_1-3Alkylene) NHC_1-3Alkyl groups; r^3aIs C_1-3An alkyl group; when R is^3aWhen the carbon atom to which it is attached forms a double bond, R^3aIs absent; r⁴Selected from H and C_1-3Alkyl groups; r^4aIs H; when R is^4aWhen the carbon atom to which it is attached forms a double bond, R^4aIs absent; each R^AIndependently selected from the group consisting of ═ O, ═ S, CN and C_1-3Alkyl radical, C_1-3Hydroxyalkyl radical, S (C)_1-3Alkyl) and C (O) OH.

In some embodiments, ring a is 5-6 membered heteroaryl, optionally with 1,2, or 3 independently selected R^AAnd (4) substituting the group. In some embodiments, ring a is 5-6 membered heterocycloalkyl, optionally substituted with 1,2, or 3 independently selected R^AAnd (4) substituting the group. In some embodiments, each R of formula III^AIndependently selected from the group consisting of ═ O, ═ S, CN, CH₃、CH₂OH、SCH₃And C (O) OH. In some embodiments, ring a is an unsubstituted 5-6 membered cycloalkyl.

In some embodiments, ring a is selected from the group consisting of:

wherein each one

Both represent a bond linking fused ring a and ring B.

In some embodiments, R of formula III¹Selected from the group consisting of H, C (O) OCH₃、OC(O)CH₃、C(S)NH₂And N-OH.

In some embodiments, R of formula III²Selected from the group consisting of H and Cl.

In some embodiments, R of formula III^2aIs H. In some embodiments, R of formula III^2aIs absent.

In some embodiments, R of formula III³Selected from H, Cl, CH₃、CH₂OH、NHC(O)CH₃And CH₂NHCH₃Group (d) of (a).

In some embodiments, R of formula III^3aIs CH₃. In some embodiments, R of formula III^3aIs absent.

In some embodiments, R of formula III⁴Selected from the group consisting of H and CH₃Group (d) of (a).

In some embodiments, the compound of formula III is selected from the group consisting of:

the present application also provides a composition comprising a compound comprising a moiety of formula IV:

and one or more pharmaceutically acceptable carriers, wherein: l is¹Selected from the group consisting of bond, C_1-3Alkylene, -O-, -O (C)_1-3Alkylene) -, C_1-3Cyanoalkylene, -S-, -SO₂-、-S(C_1-3Alkylene) -and-c (o) -or a pharmaceutically acceptable salt thereof; r¹Selected from the group consisting of phenylene, 5-6 membered heteroarylene, and 5-6 membered heterocycloalkylene, wherein each is optionally substituted with 1,2, or 3 independently selected R^ASubstituted by groups; r²Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; r³Selected from H, halogen, OH, NH₂、C(O)C_1-3Alkyl and C (S) C_1-3Alkyl groups; r⁴Selected from H, halogen, OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Haloalkyl and O (C)_1-3Cyanoalkyl); r⁵Selected from H, halogen, OH, NH₂And C (O) C_1-3Alkyl groups; r⁶Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; each R^AIndependently selected from OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Hydroxyalkyl, C (O) OH, C (O) C_1-3Alkyl and C (O) N (C)_1-3Alkyl radical)₂Wherein said C_1-3Alkyl is optionally substituted by NH₂And (4) substitution.

In some embodiments, L of formula IV¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-C (O) -in a pharmaceutically acceptable carrier.

In some embodiments, R of formula IV¹Is phenylene, optionally with 1 or 2 independently selected R^AAnd (4) substituting the group.

In some embodiments, each R of formula IV^AIndependently selected from OH, NH₂、CN、CH₃、CH₂OH、CH₂CH₂NH₂、C(O)OH、C(O)CH₃And C (O) N (CH)₃)₂Group (d) of (a).

In some embodiments, R of formula IV²Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

In some embodiments, R of formula IV³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃Group (d) of (a).

In some embodiments, R of formula IV⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN.

In some embodiments, R of formula IV⁵Selected from H, F, Cl, NH₂And C (O) CH₃Group (d) of (a).

In some embodiments, R of formula IV⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

The present application also provides methods of sensitizing and/or activating the pro-apoptotic activity of BAX comprising contacting a cell sample or tissue sample comprising BAX with a composition provided herein.

The present application further provides a method of sensitizing and/or activating the pro-apoptotic activity of BAX in a subject comprising administering to the subject a composition provided herein.

The present application further provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition provided herein.

In some embodiments, the cancer is selected from the group consisting of: breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck malignancy, breast malignancy, ovarian malignancy, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder malignancy, pancreatic malignancy, gastric cancer, colon malignancy, prostate malignancy, genitourinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical cancer, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, neuroblastoma, rhabdomyosarcoma, sarcoid sarcoma, and malignant melanoma, Kaposi's sarcoma, polycythemia vera, primary thrombocythemia, hodgkin's disease, non-hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is selected from the group consisting of: acute lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and hairy cell leukemia. In some embodiments, the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphoblastic leukemia, and chronic myelogenous leukemia.

The present application further provides a method of identifying a compound that sensitizes and/or activates the pro-apoptotic activity of a BAX polypeptide, the method comprising:

a) contacting a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with the compound in vitro; and

b) determining whether the compound binds to one or more amino acid residues selected from the group consisting of: ile80, Ala81, Ala82, Val83, Asp84, Thr85, Asp86, Ser87, Pro88, Val91, Phe116, Lys119, Leu120, Val121, Lys123, Ala124, Thr127, Leu132, and Ile 136;

wherein the binding site of the BAX polypeptide comprises a linkage of the α 3- α 4 hairpin and α 5- α 6 hairpin of the BAX polypeptide.

In some embodiments, the determining step is performed by saturation transfer difference NMR, HSQC NMR, surface plasmon resonance, biolayer interferometry, or competitive fluorescence polarization assay.

In some embodiments, binding of the compound to the BAX polypeptide results in a signal change in the NMR spectrum of the compound.

In some embodiments, the method further comprises detecting activation of the BAX polypeptide by the compound. In some embodiments, the detecting step comprises performing an assay selected from the group consisting of: detecting BAX oligomerization, antibody-based BAX conformation detection, mitochondrial cytochrome c release assay, liposome release assay, cell death assay, mitochondrial or cell morphology assay, mitochondrial calcium flux assay, mitochondrial transmembrane quantification assay, and quantification of caspase 3 activity or annexin V binding.

In some embodiments, the compound binds to the binding site with an affinity of <1 mM. In some embodiments, the compound sensitizes activation of the pro-apoptotic activity of a BAX polypeptide. In some embodiments, the compound activates the pro-apoptotic activity of a BAX polypeptide.

In some embodiments, the method further comprises administering an additional therapeutic agent that activates BAX pro-apoptotic activity. In some embodiments, the additional therapeutic agent is BIM SAHB_A2。

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; in addition, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Drawings

Figure 1a shows a BAX comprising a series of surface grooves that modulate its pro-apoptotic activity, including BH3 binding trigger and canonical sites, and inhibitory BCL-2 BH4 and vMIA interaction pockets.

Figure 1b shows a method for identifying the compounds described herein, also known as BAX-interacting fragments (BIFs), by sequential STD-NMR screening in 10, 3, single pools in sequence, yielding 56 candidate BIFs.

FIG. 1c shows that BIF-44 has no independent effect on liposomes (black, left), minimal direct BAX activation activity (black, middle), but with the addition of BIM SAHB_A2The kinetics and quantity of liposome release (black, right) were significantly enhanced later, exceeding that of BIM SAHB_A2And maximum release levels achieved by BAX combination alone (grey, right). Error bars are the mean ± SD of experiments where the technique was repeated three times and at least twice using independent liposome and protein preparations to give similar results.

FIG. 1d shows that competitive STD NMR demonstrates that BIF-44 STD signal is not susceptible to BIM SAHB_A2Effect of co-incubation.

FIGS. 2a-2b show that the liposome release assay shows little or no direct BAX activation of BIF-44 over a wide dose range, but with BIM SAHB_A2After co-incubation, direct BAX activation triggered by sensitized BH3 (fig. 2 b). Error bars are the mean ± SD of three technical replicates.

FIGS. 2c-2d show that competitive Fluorescence Polarization Assay (FPA) indicates that BIF-44 is unable to interact with FITC BIM SAHB_A2Effectively competed for BAX interaction (fig. 2c), but indeed could compete with FITC-vMIA in a dose-responsive manner (fig. 2 d). The corresponding N-terminal acetylated peptide was used as a positive control for competition in each assay: Ac-BIM SAHB_A2Blue (fig. 2 c); AcvMIA, purple (fig. 2 d). Error bars are the mean ± SD of experiments with four replicates of the technique.

FIG. 2e shows co-incubation with vmIA peptide (grey) but not with BIM SAHB_A2After co-incubation, competitive STD-NMR showed inhibition of the BIF-44 STD signal (black), consistent with the competitive FPA results shown in FIGS. 2c-2 d. Data are representative of at least two independent experiments.

FIGS. 3a-3e show the structure-activity relationship of BIF-44 analogs. Chemical structure (left), STD binding (grey) and BAX-mediated liposome release sensitizing activity of BIF-44 analogs are provided. Error bars are the mean ± SD (right) of liposome release experiments of three technical replicates. Data are representative of at least two independent experiments.

FIGS. 4a-4e show that BIF-44 targets the vmIA binding region of BAX and affects conformational kinetics.

FIG. 4a shows measurements after addition of BIF-44 (6: 1, BIF: BAX)¹⁵The most significant effects reflecting chemical shift changes above the 2SD cut-off (> 0.018ppm significance threshold) were colored red and located at the junction of the α 3- α 4 hairpin and the α 5- α 6 hairpin.A significant change at the 1SD cut-off threshold (> 0.012ppm significance threshold) was colored orange, encompassing the internal residues of the α 5 and α 6 cores and the discrete juxtaposed residues of α 1 and α 2.

FIG. 4b shows a band diagram in which residues represented by red and orange bars in the residue map of FIG. 4a are mapped accordingly to monomeric BAX (PDB ID: 1F 16). the most prominent chemical shift changes (2SD cut-off) are located in the region associated with vMIA peptide (purple) interaction.A second set of chemical shift changes (1SD cut-off) are located at the inner side-by-side residues of α 5, α 6 and α 1, α 2, suggesting allosteric sensing from the adjacent hydrophobic core to the α 1-loop- α 2 region of the BAX N terminal face.

FIG. 4c shows the results based on observations after titration of BIF-44¹⁵Molecular docking of BIF-44 with chemical shift changes of N-BAX (black, 2SD, grey 1 SD.) BIF-44 is shown on surface (left) and ribbon (middle, right) views to bind tightly to the deep cleft formed by the hydrophobic α 5 and α 6 helices of BAX and the α 3- α 4 hairpin.

FIG. 4d shows the C of each BAX residue during 100ns molecular dynamics simulation of BAX in the presence (grey) or absence (black) of BIF-44_αRMSF value of (a).

FIG. 4e shows the difference in RMSF (Δ RMSF) between the unliganded and liganded forms of BAX residues above an SD threshold are shown in grey, indicating increased mobility following BIF-44 binding and are located in the region α 1- α 2 of BAX residues from the N-terminal and C-terminal non-structural moieties (residues 1-15 and residue 188-.

FIGS. 5a-5d show that HXMS reveals allosteric deprotection of the α 1- α 2 loop and the BAX BH3 domain after BIF-44 binding.

FIG. 5a shows that addition of BIF-44 to BAX (30. mu.M, 10: 1 BIF: BAX) in a liposomal environment triggers a regiospecific increase in deuterium incorporation as measured by HXMS compared to unliganded BAX. The relative difference plot reflects the relative deuterium incorporation of BIF-44/BAX minus the relative deuterium incorporation of BAX. Dark grey shading indicates changes below the significance threshold of 0.5Da in the graph, while light grey shading and white areas highlight changes above the baseline significance threshold of 0.5Da and the stricter threshold of 0.8Da, respectively. Data are representative of at least two independent experiments.

FIG. 5b shows that the BIF-44 induced deprotection region comprises peptide fragments corresponding to amino acids 46-74, highlighted in black in the band diagram (left, PDB ID: 1F16) and amino acid sequence (SEQ ID NO: 1, right), and mapped to the key α 1- α 2 loop and BH3 region of BAX.

Figures 5c-5d show inhibition of deprotection induced by BIF-44 by co-incubation with anti-BAX BH3 antibody (figure 5c), but not with BAX6a7 antibody (figure 5d), which binds to the N-terminal residue of conformationally activated BAX. BAX amino acid sequences identified by the BAX BH3 antibody and the BAX6a7 antibody are underlined in light gray and dark gray, respectively. The relative difference plots reflect the relative deuterium incorporation of BIF-44/BAX/BH3 Ab (FIG. 5c) and BIF-44/BAX/6A7 Ab (FIG. 5d) minus the relative deuterium incorporation of BAX. Data are representative of at least two independent experiments.

FIGS. 6a-6c show the BAX conformational activation and cytochrome c release activity triggered by BIF-44-sensitized BH 3.

FIG. 6a shows BIF-44 (light grey border), BIM SAHB after exposure_A2Comparative HXMS plot of BAX in the presence of liposomes (dark grey) or after both ligands (black). The relative difference maps reflect BIF-44/BAX (light grey border), BIM SAHB_A2/BAX (dark gray) and BIF-44/BIM SAHB_A2Relative deuterium incorporation of/BAX (black) minus relative deuterium incorporation of BAX. Dark grey shading indicates changes below the significance threshold of 0.5Da in the graph, while light grey shading and white areasThe domains highlighted changes above the baseline significance threshold of 0.5Da and the stricter threshold of 0.8Da, respectively. Data are representative of at least two independent experiments.

FIG. 6b shows a band diagram (PDB ID: 1F16) and amino acid sequence (SEQ ID NO: 1) highlighted in green by using the synergistic BIF-44/BIM SAHB_A2The main regions of deprotection induced by treatment of BAX were combined (α 1, α 1- α 2 loop and α 2).

FIG. 6c shows Alb from isolation^CreBax^f/fBak^-/-BIF-44 dose-responsive sensitive BIM SAHB of mouse liver mitochondria_A2Triggered BAX-mediated cytochrome c release. Error bars are the mean ± SD of experiments where the technique was repeated three times and repeated two more times using independent preparation and treatment of mitochondria to give similar results.

FIGS. 7a-7c show STD and CPMG NMR analysis of the BIF-44/BAX interaction.

FIGS. 7a-7b show STD NMR of BIF-44 in the presence and absence of BAX protein. In the presence or absence of BAX, the off-resonance condition showed no effect on the aromatic region of BIF-44 (FIG. 7 a). The STD signal of BIF-44 was only detected in the presence of BAX (STD off-resonance minus resonance), reflecting ligand-protein interactions (fig. 7 b).

FIG. 7c shows CPMG NMR of BIF-44 in the presence and absence of BAX. Addition of BAX protein enhances the transverse relaxation rate R of BIF-44 ligand₂This is reflected by a sharp drop in the 1H-NMR signal and indicates ligand-protein interactions.

FIGS. 8a-8b show FITC-BIM SAHB_A2And vMIA peptide directly bound to BAX. Fluorescence polarization assay demonstrated BAX and FITC-BIM SAHB_A2There was a direct interaction between the peptides FITC-vMIA (FIG. 8 b). Error bars are the mean ± SD of four replicates.

FIG. 9 shows BAX after titration with BIF-44¹⁵N-HSQC analysis. And (3) adding the following components in percentage by weight of 4: 1. 6: 1 and 8: 1 (BIF: BAX) ratio measured when BIF-44 was added¹⁵The N-BAX chemical shift change is plotted as a function of the number of BAX residues. Each dose ratio (≧ 0.012, 0.018, and 0.022ppm significance threshold) The significant changes at the 1SD cut-off threshold are colored black, blue, and red, respectively.

FIG. 10 shows Isothermal Titration Calorimetry (ITC) measurements, which indicate that BIF-44 binds to BAX with a dissociation constant (KD) of 37. + -.12. mu.M. The combined raw heats were fitted to a single point combination model. BIF-44 was injected into cells containing 0.15mM BAX at a concentration of 1mM (2. mu.L per injection). The samples were diluted to the indicated concentrations in 20mM potassium phosphate buffer (pH6.2, 1% DMSO).

FIG. 11 shows BIF-44 sensitized BAX-mediated liposome release independent of the order of addition. Whether in the addition of BIM SAHB_A2While (left) adding BIM SAHB_A2Before (Right) or after addition of BIM SAHB_A2The same level of BAX activation was achieved with subsequent (intermediate) addition of BIF-44. BAX and BIF-44 were present at 750nM and 113. mu.M (150X), respectively. Samples were diluted in liposome release assay buffer (10mM HEPES, 200mM KCl, 1mM MgCl2, pH 7.0).

FIG. 12 shows BIF-44 as¹No line broadening is shown in the H-NMR spectrum, unlike the two known small molecule aggregates 4-ADPA and I4 PTH. The sample was washed with 20mM potassium phosphate buffer (pH6.2, 10% D)₂O) was diluted to the indicated concentration.

Figure 13 shows that BIF-44 does not show a rapid or dose-dependent decrease in T2 decay, whereas the aggregating compound 4-ADPA shows a rapid T2 decay (upper panel, grey). BIF-44 has a long decay time independent of concentration (lower panel). The sample was washed with 20mM potassium phosphate buffer (pH6.2, 10% D)₂O) was diluted to the indicated concentration.

Figure 14 shows dynamic light scattering indicating that BIF-44 does not aggregate in solution. Although 4-ADPA (grey) and I4PTH (black) showed a dose-dependent increase in light scattering, the BIF-44 signal remained flat. The sample was diluted to the specified concentration in 20mM potassium phosphate buffer (pH 6.2).

FIG. 15 shows global docking to define BIF-44 binding sites on BAX. BIF-44 was docked to all 20 NMR solution structures (PDB: 1F16) using HSQC results to guide docking. The pose with the best combination score for each model is displayed. The BIF-44 binding pocket (lower panel, black) consists of the following residues: ile80, Ala81, Ala82, Val83, Asp84, Thr85, Asp86, Ser87, Pro88, Val91, Phe116, Lys119, Leu120, Val121, Lys123, Ala124, Thr127, Leu132 and Ile 136.

Detailed Description

For such small proteins, a surprisingly large series of regulatory surface and complex conformational changes have been defined for BAX, as shown in figure 1 a. In its conformationally inactive state, BAX is predominantly in the cytosol, and may also be formed by anti-apoptotic proteins (e.g., BCL-X)_L) The mediated antiport process shuttles to and from the outer mitochondrial membrane (MOM) region (see, e.g., Edlichet al, Cell 2011,145: 104-116.) in response to stress, BH 3-only direct activating proteins (e.g., BIM, BID and PUMA) can directly and sequentially engage the α 1/α 6 trigger site and the typical hydrophobic groove to initiate and propagate BAX homo-oligomerization reactions (see, e.g., Czabotar et al, Cell 2013,152: 519-531; Edwards et al, chem. biol.2013,20: 888-902; Gavathitis et al, mol. Cell 2010,40: 481-492; Gavathitis et al, Nature,2008,455: 6-1081), while BCL-2 canonical groove, BCL-2 BH4 and cytomegalovirus proteins can bind to and inhibit BAX (see, e.g., Scotchl. 3019, Nature, 1076-19. 11. wo-11. sub.05, a conformational changes within the basic mitochondrial membrane of the BAX-2-Bax motif, such as Biol et al, 103-120, 103-52-11-19, and the constitutive transport mechanisms of BCL-2-BH-2-19, such as-2-t-mediated apoptosis-mediated constitutive, intracellular, and-mediated constitutive mechanisms, e.7-mediated apoptosis-mediated constitutive, e.7-mediated constitutive, e.g., intracellular, e.g, e.7-mediated constitutive, e.g., intracellular.

Given the central role of BCL-2 family proteins in apoptosis regulation during health and disease, efforts are underway to abrogate the role of anti-apoptotic proteins in cancer, where sequestration and inactivation of pro-apoptotic members promotes cell immortalization. In particular, this mechanism by which anti-apoptotic proteins (e.g., BCL-2) deploy surface grooves to capture the apoptotic trigger BCL-2 homolog 3(BH3) helix of pro-apoptotic proteins has now been used to make venetochlax,a selective BCL-2 pocket inhibitor (see, e.g., Souers et al, Nat. Med.2013,19: 202-98208; Sattler et al, Science,1997,275: 983-986). This therapeutic strategy of "inhibiting said inhibitors" is being used to develop drugs against a broad spectrum of anti-apoptotic targets involved in cancer, including BCL-X_L(see, e.g., Lessene et al, Nat. chem. biol.2013,9: 390-.

It has been found that the α 1/α 6 trigger site of BAX is directly activated by the pro-apoptotic BH3 domain, and it is believed that driving cancer cell death with the "activation of the activator" strategy would also merit therapeutic exploration (see, e.g., Gavathiotis et al, mol. cell,2010,40: 481-492; Gavathiotis et al, Nature,2008,455: 1076-1081). this work was previously reported to be initiated by bioinformatic screening, since the production of BAX for direct experimental screening was challenged by the expression of sufficient amounts of recombinant BAX and the general instability of BAX in solution, especially when exposed to potential activators, bioinformatics and subsequent biochemical and cell validation work produced the first directly selective BAX Activator Molecule (BAM) (see, e.g., gavatios et al, nat. chem. biol.2012,8: 639.) this application was directed to the development of a small nuclear magnetic resonance-mediated activation compound by the previous nuclear magnetic resonance screening and provides a potential nuclear magnetic resonance for the development of BAX compound.

Thus, the present application provides compounds or molecular fragments that bind to BAX at the deep hydrophobic pocket in a region that may otherwise be naturally occluded by the BAX inhibitory BH4 domain of BCL-2 (see, e.g., Barclay et al, mol.cell,2015,57: 873-containing 886) or cytomegalovirus vMIA polypeptide (see, e.g., Ma et al, proc.natl.acad.sci.u.s.a.2012,109: 20901-containing 20906.) additionally, the present application describes mobilization of BAX by molecular ligation through the α 1- α 2 loop and the variation of the BAX BH3 helix highlights key mechanistic steps involved in direct activation and homooligomerization of BAX mediated by BH3 (see, e.g., gavatithios et al, mol.cell, cell, 40: 481-containing 492; Wang et al, mol.cell. biol.1998,18: 6089).

Compounds and compositions

The present application provides, inter alia, compositions comprising: a compound of formula I or a pharmaceutically acceptable salt thereof

And one or more pharmaceutically acceptable carriers, wherein, L¹Selected from the group consisting of bond, C_1-3Alkylene, -O-, -O (C)_1-3Alkylene) -, C_1-3Cyanoalkylene, -S-, -SO₂-、-S(C_1-3Alkylene) -and-c (o) -or a pharmaceutically acceptable salt thereof; r¹Selected from halogen, OH, C_1-3Alkyl radical, C_1-3Haloalkyl, NH₂CN, phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein said phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl are each optionally substituted with 1,2 or 3 independently selected R^ASubstituted by groups; r²Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; r³Selected from H, halogen, OH, NH₂、C(O)C_1-3Alkyl and C (S) C_1-3Alkyl groups; r⁴Selected from H, halogen, OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Haloalkyl and O (C)_1-3Cyanoalkyl); r⁵Selected from H, halogen, OH, NH₂And C (O) C_1-3Alkyl groups; r⁶Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; each R^AIndependently selected from OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Hydroxyalkyl, C (O) OH, C (O) C_1-3Alkyl and C (O) N (C)_1-3Alkyl radical)₂Wherein said C_1-3Alkyl is optionally substituted by NH₂And (4) substitution.

In some embodiments, L of formula I¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-C (O) -in a pharmaceutically acceptable carrier.

In some embodiments, L of formula I¹is-O-, -CH₂-or-OCH₂-。

In some embodiments, R of formula I¹Is phenyl, optionally substituted by 1 or 2 independently selected R^AAnd (4) substituting the group.

In some embodiments, R of formula I¹Is phenyl, optionally substituted by 1 or 2 independently selected R^AIs substituted by a group, wherein each R^AIndependently selected from OH, NH₂、CH₂OH and C (O) OH.

In some embodiments, R of formula I¹Is phenyl, 4-hydroxyphenyl, 3-hydroxyphenyl, 4-aminophenyl, 4-carboxyphenyl or 4-hydroxymethylphenyl.

In some embodiments, R of formula I²Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

In some embodiments, R of formula I²Is H or CH₃。

In some embodiments, R of formula I³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃Group (d) of (a).

In some embodiments, R of formula I³Is H.

In some embodiments, R of formula I⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN.

In some embodiments, R of formula I⁴Is H or OH.

In some embodiments, R of formula I⁵Selected from H, F, Cl, NH₂And C (O) CH₃Group (d) of (a).

In some embodiments, R of formula I⁵Is H or NH₂。

In some embodiments, R of formula I⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

In some embodiments, R of formula I⁶Is H.

In some embodiments, L of formula I¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-c (o) -and; r of the formula I¹Selected from the group consisting of Cl, CH₃、CF₃、NH₂CN, phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl are each optionally substituted with 1 or 2 independently selected R^ASubstituted by groups; each R of the formula I^AIndependently selected from OH, NH₂、CN、CH₃、CH₂OH、CH₂CH₂NH₂、C(O)OH、C(O)CH₃And C (O) N (CH)₃)₂A group of (a); r of the formula I²Selected from H, Cl, CN, CH₃And C (O) OCH₃A group of (a); r of the formula I³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃A group of (a); r of the formula I⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN; r of the formula I⁵Selected from H, F, Cl, NH₂And C (O) CH₃A group of (a); r of the formula I⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

In some embodiments, L of formula I¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-c (o) -and; r of the formula I¹Is phenyl, optionally substituted by 1 or 2 independently selected R^ASubstituted by groups; each R of the formula I^AIndependently selected from OH, NH₂、CN、CH₃、CH₂OH、CH₂CH₂NH₂、C(O)OH、C(O)CH₃And C (O) N (CH)₃)₂A group of (a); r of the formula I²Selected from H, Cl, CN, CH₃And C (O) OCH₃A group of (a); r of the formula I³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃A group of (a); r of the formula I⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN; r of the formula I⁵Selected from H, F, Cl, NH₂And C (O) CH₃A group of (a); r of the formula I⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

in some embodiments, the compound of formula I is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula I is:

or a pharmaceutically acceptable salt thereof.

The present application also provides a composition comprising: a compound of formula II or a pharmaceutically acceptable salt thereof

And one or more pharmaceutically acceptable carriers, wherein, X¹Is NH or S; x²Is C or N; l is¹Selected from the group consisting of a bond, -C (O) -, -C (O) O-and-SO₂-a group of compositions; r¹Selected from the group consisting of C_1-3Alkyl, NH₂Two (C)_1-3Alkyl) amino and 5-6 membered heterocycloalkyl; r²Selected from H, halogen, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; r³Selected from the group consisting of H, C_1-3Alkyl and 5-6 membered heteroaryl; when X is present²When is N, R³Is absent; r⁴Selected from H and C_1-3Alkyl groups.

In some embodiments, X of formula II¹Is NH.

In some embodiments, X of formula II¹Is S.

In some implementationsIn scheme, X of formula II²Is C.

In some embodiments, X of formula II²Is N.

In some embodiments, X of formula II¹Is NH, and X of the formula II²Is C.

In some embodiments, X of formula II¹Is NH, and X of the formula II²Is N.

In some embodiments, X of formula II¹Is S, and X of the formula II²Is C.

In some embodiments, X of formula II¹Is S, and X of the formula II²Is N.

In some embodiments, R of formula II¹Is selected from the group consisting of CH₃、CH₂CH₃、NH₂、N(CH₂CH₃)₂Piperidinyl and dihydrothiophen-3 (2H) -onyl.

In some embodiments, R of formula II⁴Selected from H and C_1-3Alkyl groups.

In some embodiments, X of formula II¹Is NH or S; x of the formula II²Is C or N; l of the formula II¹Selected from the group consisting of a bond, -C (O) -, -C (O) O-and-SO₂-a group of compositions; r of the formula II¹Is selected from the group consisting of CH₃、CH₂CH₃、NH₂、N(CH₂CH₃)₂Piperidinyl and dihydrothiaA thiophen-3 (2H) -keto group; r of the formula II²Selected from H, Cl, CH₃And C (O) OCH₂CH₃A group of (a); r of the formula II³Selected from the group consisting of H, CH₃、CH₂CH₃And thienyl; r of the formula II⁴Selected from H and C_1-3Alkyl groups.

In some embodiments, X of formula II¹Is NH or S; x of the formula II²Is C or N; -L of the formula II¹-R¹Form a radical selected from the group consisting of NH₂、C(O)OCH₃、C(O)OCH₂CH₃、C(O)N(CH₂CH₃)₂、SO₂-a group of the group consisting of piperidinyl and dihydrothiophen-3 (2H) -onyl; r of the formula II²Selected from H, Cl, CH₃And C (O) OCH₂CH₃A group of (a); r of the formula II³Selected from the group consisting of H, CH₃、CH₂CH₃And thienyl; r of the formula II⁴Selected from H and C_1-3Alkyl groups.

the present application also provides a composition comprising: a compound of formula III or a pharmaceutically acceptable salt thereof

And one or more pharmaceutically acceptable carriers, wherein,

a single bond or a double bond; ring A is selected from the group consisting of 5-6 membered cycloalkyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, forming a fused ring with ring B, wherein ring A is optionally substituted with 1,2 or 3 independently selected R^ASubstituted by groups; r¹Selected from the group consisting of H, C(O)OC_1-3Alkyl, OC (O) C_1-3Alkyl, C (S) NH₂And ═ N-OH; r^1aIs H; when R is^1aWhen the carbon atom to which it is attached forms a double bond, R^1aIs absent; r²Selected from the group consisting of H and halogen; r^2aIs H; when R is^2aWhen the carbon atom to which it is attached forms a double bond, R^2aIs absent; r³Selected from H, halogen, C_1-3Alkyl radical, C_1-3Hydroxyalkyl, NHC (O) C_1-3Alkyl and (C)_1-3Alkylene) NHC_1-3Alkyl groups; r^3aIs C_1-3An alkyl group; when R is^3aWhen the carbon atom to which it is attached forms a double bond, R^3aIs absent; r⁴Selected from H and C_1-3Alkyl groups; r^4aIs H; when R is^4aWhen the carbon atom to which it is attached forms a double bond, R^4aIs absent; each R^AIndependently selected from the group consisting of ═ O, ═ S, CN and C_1-3Alkyl radical, C_1-3Hydroxyalkyl radical, S (C)_1-3Alkyl) and C (O) OH.

In some embodiments, ring a is 5-6 membered heteroaryl, optionally with 1,2, or 3 independently selected R^AAnd (4) substituting the group.

In some embodiments, ring a is 5-6 membered heterocycloalkyl, optionally substituted with 1,2, or 3 independently selected R^AAnd (4) substituting the group.

In some embodiments, each R of formula III^AIndependently selected from the group consisting of ═ O, ═ S, CN, CH₃、CH₂OH、SCH₃And C (O) OH.

In some embodiments, ring a is an unsubstituted 5-6 membered cycloalkyl.

In some embodiments, ring a is selected from the group consisting of:

wherein each one

Both represent a bond linking fused ring a and ring B.

In some embodiments, R of formula III^2aIs H.

In some embodiments, R of formula III^2aIs absent.

In some embodiments, R of formula III^3aIs CH₃。

In some embodiments, R of formula III^3aIs absent.

In some embodiments, ring a is selected from the group consisting of 5-6 membered heteroaryl, 5-6 membered heterocycloalkyl, and unsubstituted 5-6 membered cycloalkyl, wherein the 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl are each optionally substituted with 1,2, or 3 independently selected R^ASubstituted by groups; r of the formula III¹Selected from the group consisting of H, C (O) OCH₃、OC(O)CH₃、C(S)NH₂And ═ N-OH; r of the formula III²Selected from the group consisting of H and Cl; r of the formula III^2aIs H; when R is^2aWhen the carbon atom to which it is attached forms a double bond, R^2aIs absent; r of the formula III³Selected from H, Cl, CH₃、CH₂OH、NHC(O)CH₃And CH₂NHCH₃A group of (a); r of the formula III^3aIs C_1-3An alkyl group; when R is^3aAttached carbon atom formWhen forming a double bond, R^3aIs absent; r of the formula III⁴Selected from the group consisting of H and CH₃A group of (a); r of the formula III^4aIs H; when R is^4aWhen the carbon atom to which it is attached forms a double bond, R^4aIs absent; each R of the formula III^AIndependently selected from the group consisting of ═ O, ═ S, CN, CH₃、CH₂OH、SCH₃And C (O) OH.

the present application also provides a composition comprising a compound comprising a moiety of formula IV, or a pharmaceutically acceptable salt thereof,

and one or more pharmaceutically acceptable carriers, wherein, L¹Selected from the group consisting of bond, C_1-3Alkylene, -O-, -O (C)_1-3Alkylene) -, C_1-3Cyanoalkylene, -S-, -SO₂-、-S(C_1-3Alkylene) -and-c (o) -or a pharmaceutically acceptable salt thereof; r¹Selected from the group consisting of phenylene, 5-6 membered heteroarylene, and 5-6 membered heterocycloalkylene, wherein each is optionally substituted with 1,2, or 3 independently selected R^ASubstituted by groups; r²Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; r³Selected from H, halogen, OH, NH₂、C(O)C_1-3Alkyl and C (S) C_1-3Alkyl groups; r⁴Selected from H, halogen, OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Haloalkyl and O (C)_1-3Cyanoalkyl) groupGroup (b); r⁵Selected from H, halogen, OH, NH₂And C (O) C_1-3Alkyl groups; r⁶Selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups; each R^AIndependently selected from OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Hydroxyalkyl, C (O) OH, C (O) C_1-3Alkyl and C (O) N (C)_1-3Alkyl radical)₂Wherein said C_1-3Alkyl is optionally substituted by NH₂And (4) substitution.

In some embodiments, R of formula IV¹Is phenylene optionally substituted with 1 or 2 independently selected R^AAnd (4) substituting the group.

In some embodiments, L of formula IV¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-c (o) -and; r of the formula IV¹Is phenylene, optionally substituted with 1 or 2 independently selected R^ASubstituted by groups; each R of the formula IV^AIndependently selected from OH, NH₂、CN、CH₃、CH₂OH、CH₂CH₂NH₂、C(O)OH、C(O)CH₃And C (O) N (CH)₃)₂A group of (a); r of the formula IV²Selected from the group consisting of H, Cl, CN, CH3 and C (O) OCH₃A group of (a); r of the formula IV³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃A group of (a); r of the formula IV⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN; r of the formula IV⁵Selected from H, F, Cl, NH₂And C (O) CH₃A group of (a); r of the formula IV⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

When used as a medicament, the compositions provided herein may be administered in the form of a pharmaceutical composition. These compositions may be prepared as described herein or elsewhere and may be administered by various routes depending on whether local or systemic treatment is desired and the area to be treated. Can be administered topically (including transdermal, epidermal, ocular and mucosal (including intranasal, vaginal and rectal delivery)), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), orally or parenterally. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular injection or intraperitoneal intramuscular infusion; or intracranial (e.g., intrathecal or intracerebroventricular) administration. Parenteral administration may be in the form of a single bolus dose, or may be, for example, by continuous infusion pump.

Pharmaceutical compositions for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers; an aqueous, powder or oily base; thickeners and the like may be necessary or desirable.

Also provided are compositions comprising a compound provided herein (e.g., a compound of formulas I-III or a compound comprising a moiety of formula IV) or a pharmaceutically acceptable salt thereof as an active ingredient, in combination with one or more pharmaceutically acceptable carriers (e.g., excipients). In preparing the compositions provided herein, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier, for example, in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material that serves as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or liquid medium), ointments, soft and hard gel capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The composition may additionally include, but is not limited to, lubricants such as talc, magnesium stearate, and mineral oil; a wetting agent; emulsifying and suspending agents; preservatives, such as methyl benzoate and propyl hydroxybenzoate; a sweetener; a flavoring agent, or a combination thereof.

The active ingredient may be effective over a wide dosage range and is generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the compound and/or composition actually administered will generally be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound or composition administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

In various places in the specification, divalent linking substituents are described. It is specifically intended that each divalent linking substituent comprises a forward form and a reverse form of the linkageAnd (4) a substituent. For example, -NR (CR' R ")_n-comprises-NR (CR 'R')_n-and- (CR 'R')_nNR-. If the structure explicitly requires a linking group, the markush variables listed for that group should be understood as linking groups.

As used herein, the phrase "optionally substituted" means unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced with a substituent. It is understood that substitution on a given atom is limited by valence.

In all definitions, the term "C_n-m"denotes a range including endpoints, where n and m are integers and represent a carbon number. Examples include C_1-4、C_1-6And the like.

As used herein, the term "C" used alone or in combination with other terms (e.g., cyanoalkylene)_n-mAlkylene "refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, methylene, ethylene-1, 2-diacyl (ethane-1, 2-diyl), propylene-1, 3-diacyl (propan-1,3-diyl), propylene-1, 2-diacyl, and the like. In some embodiments, the alkylene moiety comprises 1 to 3 carbon atoms or 1 to 2 carbon atoms.

As used herein, the term "C_n-mCyanoalkylene "refers to a divalent alkyl linking group having n to m carbons, wherein the alkyl linking group is substituted with one or more cyano (i.e., -CN) groups. In some embodiments, cyanoalkylene contains 1 cyano group.

As used herein, the term "C" used alone or in combination with other terms_n-mAlkyl "refers to a saturated hydrocarbon group having n to m carbons that may be straight or branched. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl; higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2, 2-trimethylpropyl and the like. In some embodiments, the alkyl group contains 1 to 3 carbon atoms or 1 to 2 carbon atoms.

As used herein, "halogen" refers to F, Cl, Br, or I. In some embodiments, the halogen is F, Cl or Br. In some embodiments, halogen is F or Cl.

As used herein, the term "C" used alone or in combination with other terms_n-mHaloalkyl "refers to an alkyl group having one halogen atom to 2s +1 halogen atoms (which may be the same or different), where" s "is the number of carbon atoms in the alkyl group, where the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group comprises 1 to 3 carbon atoms or 1 to 2 carbon atoms. In some embodiments, the haloalkyl group contains 1 halo group.

As used herein, the term "C" used alone or in combination with other terms_n-mHydroxyalkyl "refers to an alkyl group having one hydroxyl group (i.e., -OH) to 2s +1 hydroxyl groups, where" s "is the number of carbon atoms in the alkyl group, where the alkyl group has n to m carbon atoms. In some embodiments, the hydroxyalkyl group comprises 1 to 3 carbon atoms or 1 to 2 carbon atoms. In some embodiments, the hydroxyalkyl group contains 1 hydroxyl group.

As used herein, the term "C" used alone or in combination with other terms_n-mCyanoalkyl "refers to an alkyl group having one cyano group (i.e., -CN) to 2s +1 cyano groups, where" s "is the number of carbon atoms in the alkyl group, where the alkyl group has n to m carbon atoms. In some embodiments, the cyanoalkyl group contains 1 to 3 carbon atoms or 1 to 2 carbon atoms. In some embodiments, the cyanoalkyl group contains 1 cyano group.

As used herein, the term "di (C)_n-mBy alkyl) amino is meant the formula-N (alkyl)₂Wherein the two alkyl groups each independently have n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 3 carbon atoms or 1 to 2 carbon atoms.

As used herein, "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group that includes cyclic alkyl and/or alkenyl groups. Cycloalkyl groups may include monocyclic or polycyclic (e.g.Having 2,3 or 4 fused rings) groups and spirocycles. Cycloalkyl groups may have 3,4, 5 or 6 ring-forming carbons (i.e., C)_3-6Cycloalkyl groups). The ring-forming carbon atoms of the cycloalkyl group may be optionally substituted with oxo or thioxo (e.g., ═ O or ═ S). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. In some embodiments, cycloalkyl has 3-6 ring-forming carbon atoms (i.e., C)_3-6A cycloalkyl group).

As used herein, the term "heteroaryl" refers to an aromatic monocyclic or polycyclic heterocyclic ring having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1,2,3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl group has 5 to 6 ring atoms and 1,2,3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. Exemplary five-membered ring heteroaryls include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2, 3-triazolyl, tetrazolyl, 1,2, 3-thiadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-triazolyl, 1,2, 4-thiadiazolyl, 1,2, 4-oxadiazolyl, 1,3, 4-triazolyl, 1,3, 4-thiadiazolyl, and 1,3, 4-oxadiazolyl. Exemplary six-membered ring heteroaryls include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

As used herein, the term "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having 1,2,3, or 4 ring-forming heteroatoms selected from O, N or S. Included in heterocycloalkyl are monocyclic 4-, 5-and 6-membered heterocycloalkyl groups. Exemplary heterocycloalkyl groups include pyranyl, oxetanyl, azetidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. The ring-forming carbon atoms and heteroatoms of the heterocycloalkyl group may be optionally substituted by oxo or thioxo (e.g., ═ O, ═ S). The heterocycloalkyl group may be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some implementationsIn embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties having one or more aromatic rings fused (i.e., having a common bond) to a cycloalkyl ring, e.g., piperidine, morpholine, aza

And the like, benzo-or thienyl-derivatives. The heterocycloalkyl group comprising a fused aromatic ring can be attached through any ring-forming atom including ring-forming atoms of the fused aromatic ring. In some embodiments, heterocycloalkyl has 5-6 ring atoms with 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

As used herein, the term "phenylene" refers to a divalent phenyl linking group.

As used herein, the term "heteroarylene" refers to a divalent heteroaryl linking group. In some embodiments, the heteroarylene group has 5-6 ring atoms.

As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkyl linking group. In some embodiments, heterocycloalkylene has 5-6 ring atoms.

In certain places, definitions or embodiments refer to particular rings (e.g., azetidine rings, pyridine rings, etc.). Unless otherwise indicated, the rings may be attached to any ring member so long as the valency of the atoms is not exceeded. For example, the azetidine ring may be attached at any position on the ring, while the pyridin-3-yl ring is attached at the 3-position.

The term "compound" as used herein is meant to include all stereoisomers, geometric isomers, tautomers and isotopes of the structures described. Unless otherwise indicated, a compound identified herein by name or structure as one particular tautomeric form is intended to include other tautomeric forms.

The compounds provided herein also include tautomeric forms. The tautomeric form results from the exchange of a single bond with an adjacent double bond and the concomitant migration of protons. Tautomeric forms include prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge. Typical prototropic tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons may occupy two or more positions of a heterocyclic ring system, such as 1H-and 3H-imidazole, 1H-, 2H-and 4H-1,2, 4-triazole, 1H-and 2H-isoindole, and 1H-and 2H-pyrazole. Tautomeric forms can be in equilibrium by appropriate substitution, or sterically locked into one form.

The term "pharmaceutically acceptable" as used herein refers to those compounds, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by conversion of an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; acidic residues such as basic or organic salts of carboxylic acids, and the like. Pharmaceutically acceptable salts herein include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, alcohols (e.g. methanol, ethanol, isopropanol or butanol) or acetonitrile (MeCN) are preferred. A list of suitable salts can be found in Remington's Pharmaceutical sciences,17th ed., Mack Publishing Company, Easton, Pa.,1985, p.1418 and journal of Pharmaceutical Science,66,2 (1977). Conventional methods for preparing salt forms are described, for example, in handbook of Pharmaceutical Salts, Properties, Selection, and Use, Wiley-VCH, 2002.

Application method

The present application further provides a method of sensitizing and/or activating the pro-apoptotic activity of BAX. In some embodiments, the method comprises contacting a cell sample or tissue sample comprising BAX with a composition provided herein (e.g., a composition comprising a compound of formulae I-III or a compound comprising a moiety of formula IV, or a pharmaceutically acceptable salt thereof). As used herein, the term "contacting" refers to bringing together specified components in an in vitro system. For example, "contacting" a BAX polypeptide with a composition provided herein includes introducing a compound of the invention into a sample (e.g., a cell sample or tissue sample) comprising cells or a purified preparation containing the BAX polypeptide. In some embodiments, a composition comprising a compound of formulae I-III or a compound comprising a moiety of formula IV sensitizes in a cell sample or tissue sample the pro-apoptotic activity of a BAX polypeptide activated by another pro-apoptotic agent (i.e., enhances the pro-apoptotic activity of a BAX polypeptide induced by the pro-apoptotic agent). In such embodiments, the compositions described herein may or may not themselves activate the pro-apoptotic activity of BAX polypeptides. In some embodiments, a composition comprising a compound of formulae I-III or a compound comprising a moiety of formula IV activates the pro-apoptotic activity of a BAX polypeptide in a cell sample or a tissue sample. In such embodiments, the composition may be administered in the presence or absence of another pro-apoptotic agent.

In some embodiments, the present application provides a method of sensitizing and/or activating the pro-apoptotic activity of BAX in a subject. In some embodiments, the method comprises administering to the subject a compound or composition provided herein. In some embodiments, a compound or composition provided herein sensitizes activation of pro-apoptotic activity of BAX in a subject (e.g., when the composition is administered in combination with another pro-apoptotic agent). In some embodiments, a compound or composition provided herein activates the pro-apoptotic activity of BAX in a subject (e.g., when the composition is administered in the presence or absence of another pro-apoptotic agent). As used herein, the term "subject" refers to any animal, including mammals. Examples of subjects include, but are not limited to, mice, rats, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition provided herein. As used herein, the phrase "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that a researcher, veterinarian, medical doctor or other clinician seeks in a tissue, system, animal, individual, or human.

The present application further provides a method of treating cancer in a subject. In some embodiments, the methods comprise administering to a subject in need of such treatment a therapeutically effective amount of a composition provided herein.

Exemplary cancers include, but are not limited to: breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck malignancy, breast malignancy, ovarian malignancy, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder malignancy, pancreatic malignancy, gastric cancer, colon malignancy, prostate malignancy, genitourinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical cancer, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, neuroblastoma, rhabdomyosarcoma, sarcoid sarcoma, and malignant melanoma, Kaposi's sarcoma, polycythemia vera, primary thrombocythemia, hodgkin's disease, non-hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

Exemplary leukemias and lymphomas include, but are not limited to: erythroleukemia, acute megakaryocytic leukemia, acute lymphocytic leukemia, acute promyelocytic leukemia (APML), acute myelocytic leukemia, Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Acute Lymphoblastic Leukemia (ALL) (e.g., B-lineage ALL and T-lineage ALL), Chronic Lymphocytic Leukemia (CLL), chronic myelocytic leukemia, prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), Waldenstrom's Macroglobulinemia (WM), non-Hodgkin's lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic Leukemia (LGF), Hodgkin's disease, and Reed-Sterberg disease.

In some embodiments, the leukemia is selected from the group consisting of: acute lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

In some embodiments, the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphoblastic leukemia, and chronic myelogenous leukemia.

The application further provides methods for identifying compounds that activate the pro-apoptotic activity of BAX polypeptides. In some embodiments, the method comprises:

a) (ii) contacting the polypeptide comprising the amino acid sequence of SEQ id no: 1 with a compound in vitro; and

b) determining whether the compound binds to one or more amino acid residues selected from the group consisting of: ile80, Ala81, Ala82, Val83, Asp84, Thr85, Asp86, Ser87, Pro88, Val91, Phe116, Lys119, Leu120, Val121, Lys123, Ala124, Thr127, Leu132, Ile 136;

In some embodiments, the method further comprises detecting activation of the BAX polypeptide by the compound.

In some embodiments, the detecting step comprises performing an assay selected from the group consisting of: detecting BAX oligomerization, antibody-based BAX conformation detection, mitochondrial cytochrome c release assay, liposome release assay, cell death assay, mitochondrial or cell morphology assay, mitochondrial calcium flux assay, mitochondrial transmembrane quantification assay, and quantification of caspase 3 activity or annexin V binding.

In some embodiments, the compound binds to the binding site with an affinity of < 1mM, e.g., < 750nM, < 500nM, < 250nM, < 100nM, < 50nM, < 25nM, < 10nM, etc.

In some embodiments, the methods provided herein further comprise administering one or more additional therapeutic agents (e.g., chemotherapeutic agents) and/or performing one or more additional medical techniques (e.g., radiation therapy, surgery, etc.) to the subject, the in vitro cell sample, the tissue sample, and/or the organ sample.

In some embodiments, the methods further comprise administering one or more additional therapeutic agents selected from the group consisting of an apoptosis-inducing agent, a polynucleotide (e.g., antisense, ribozyme, siRNA), a polypeptide (e.g., enzymes and antibodies), a biomimetic (e.g., BH3 mimetic), an agent that binds to and inhibits an anti-apoptotic protein (e.g., an agent that inhibits anti-apoptotic BCL-2 protein), an alkaloid, an alkylating agent, an anti-tumor antibiotic, an antimetabolite, a hormone, a platinum compound, a monoclonal or polyclonal antibody (e.g., an antibody conjugated to an anti-cancer drug, a toxin, a defensin, etc.), a toxin, a radionuclide, a biological response modifier (e.g., an interferon, such as IFN- α, etc.) and an interleukin (e.g., IL-2, etc.), an adoptive immunotherapy agent, a hematopoietic growth factor, an agent that induces differentiation of tumor cells (e.g., all-trans retinoic acid, etc.), a gene therapy agent (e.g., an antisense therapy agent and a nucleotide), a tumor production inhibitor, a kappa protein body inhibitor, NF β modulator, an anti-HDAC compound, a CDK inhibitor, and.

In some embodiments, the methods further comprise administering one or more additional therapeutic agents (e.g., agents that inhibit anti-apoptotic BCL-2 proteins) that bind to and inhibit anti-apoptotic proteins such as ABT-263, obatoclax, gossypol derivatives, IAP inhibitors, and immobilized peptides that target anti-apoptotic proteins (e.g., MCL-1 SAHB, BID SAHB, BAD SAHB, bimahb, etc.).

In some embodiments, the method further comprises administering one or more additional therapeutic agents (e.g., pro-apoptotic agents) that bind to and activate BAX (e.g., BIM SAHB)_A2) The pro-apoptotic activity of (a). Other examples of compounds that bind to and activate the pro-apoptotic activity of BAX may be found in, for example, U.S. patent nos. 9,303,024; U.S. patent publication No. US 2016-0171150; gavatithis et al, nat. chem.biol.2012,8: 639-; brahmbhatt et, biochem.J.2016,473: 1073-1083; xin et al, nat. Commun.2014,5: 4935; and Zhao et al, mol.cell.biol.2014,34: 1198-; the disclosure of each of which is incorporated herein by reference in its entirety.

In some embodiments, the method further comprises administering one or more additional therapeutic agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); kinase inhibitors (e.g., Epidermal Growth Factor Receptor (EGFR) kinase inhibitors, Vascular Growth Factor Receptor (VGFR) kinase inhibitors, Fibroblast Growth Factor Receptor (FGFR) kinase inhibitors, platelet-derived growth factor receptor (PDGFR) kinase inhibitors, and Bcr-Abl kinase inhibitors, such as GLEEVEC); an antisense molecule; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN and AVASTIN); antiestrogens (e.g., raloxifene and tamoxifen); antiandrogens (e.g., flutamide, bicalutamide, finasteride, aminoglutamine, ketoconazole, and corticosteroids); cyclooxygenase 2(COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory agents (e.g., phenylbutazone, decaduron, DELTASONE, dexamethasone hydrosol, DEXONE, hexdol, hydroxychloroquine, METICORTEN, oradex, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-11, Fludarabine (FLUDARA), Dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE, or TAXOL); a cell signaling molecule; ceramide and cytokine, staurosporine, and the like.

In some embodiments, the subject is a subject in need thereof (e.g., a subject identified as in need of such treatment, e.g., a subject having or at risk of having one or more diseases provided herein). Identifying a subject in need of such treatment can be judged by the subject or a health care professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

In some embodiments, the subject has not previously undergone chemotherapy. In some embodiments, the subject does not have, or is not at risk of, thrombocytopenia, such as that resulting from chemotherapy, radiation therapy, or bone marrow transplantation due to treatment for cancer or lymphoma.

In some embodiments, the additional therapeutic agent is administered simultaneously with, before, or after administration of the composition provided herein. In some embodiments, the compositions provided herein are administered during a surgical procedure. In some embodiments, the compositions provided herein are administered in combination with an additional therapeutic agent during surgery.

As used herein, the term "treating" refers to one or more of the following: (1) inhibiting the disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing or exhibiting a pathology or symptomatology of the disease, condition, or disorder (i.e., arresting further development of the pathology and/or symptomatology); (2) improving the disease; for example, ameliorating a disease, condition, or disorder (i.e., reversing the pathology and/or symptomatology) in an individual who is experiencing or exhibiting the pathology or symptomatology of the disease, condition, or disorder, e.g., reducing the severity of the disease or alleviating or relieving one or more symptoms of the disease. In some embodiments, such terms refer to one, two, three, or more of the following outcomes following administration of one or more therapies: (1) stabilization, reduction, or elimination of the cancer cell population, (2) prolonging cancer remission, (3) reducing the recurrence rate of cancer, (4) prolonging cancer recurrence time, and (6) improving patient survival.

Examples

The present invention will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize that various non-critical parameters may be changed or modified to produce substantially the same results.

Example 1 peptide Synthesis

Solid state peptide synthesis was performed using Fmoc chemistry as previously described (see, e.g., Bird et al, methods enzymol.2008,446: 369-. Mixing vmIA (a)¹³¹EALKKALRRHRFLWQRRQRA¹⁵⁰-CONH₂) (SEQ ID NO:2) and BIM SAHB_A2(¹⁴⁵Ac-EIWIAQELRXIGDXFNAYYA¹⁶⁴-CONH2, X ═ binding amino acids) (SEQ ID NO:3) peptide N-terminally derivatized with acetyl or Fluorescein Isothiocyanate (FITC) - β -alanine for indicated use in NMR and biochemical experiments₆Reconstituted and diluted into the indicated aqueous buffer for experimental use.

Example 2 expression and purification of full-Length BAX

Recombinant full-length BAX was expressed in BL21(DE3) E.coli using the pTYB1 vector (see, e.g., Suzuki et al, Cell,2000,103: 645-. The cell pellet was resuspended in 20mM Tris, 250mM NaCl, pH 7.2 and lysed by two passes through a microfluidizer (Microfluidics) cooled to 4 ℃. The lysate was clarified by centrifugation at 20,000 rpm. BAX was purified by batch affinity binding using chitin resin (NewEngland Biolabs) at 4 ℃, then loaded into a gravity flow column for washing and elution. The intein-chitin binding domain tag was cleaved by incubation in 50mM dithiothreitol for 36 hours at 4 ℃. Pure protein was isolated by size exclusion chromatography (Superdex 7510/300; 20mM potassium phosphate, pH6.2) using FPLC system (GE Healthcare Life Sciences).

Example 3 fragment screening by STD-NMR

Ro3 diversity Compound library was purchased from Maybridge, Inc¹H-NMR characterization, then split into 10 groups to minimize spectral overlap. Forty compounds were excluded prior to screening as part of quality control measures to identify poorly performing compounds. The fragment pool was added to 5. mu.M unlabeled full-length human BAX solution (in 20mM potassium phosphate buffer, pH6.2, 10% (v/v) D₂O) final compound concentration was 300 μ M. The samples were mixed and loaded into 5mm NMR tubes using a liquid handling robot (Gilson). STD-NMR measurements were performed on a Varian Inova500-MHz spectrometer equipped with a helium cooled cryotip at 25 ℃. Low power saturation of the protein can be achieved by a series of 50ms gaussian pulses lasting a total of 3 seconds; resonant irradiation was performed at 0.8ppm and non-resonant irradiation was performed at 30 ppm. Standard excitation engraving was used for solvent inhibition. Each experiment was carried out for 14 minutes. The results were preliminary analyzed to determine the presence of binders (binders) by comparing the resonance STD and non-resonance STD spectra of each pool, 37 of the 96 pools showing evidence of protein interactions. Each pool was then analyzed using internal display analysis and display software to identify individual binders, allowing accurate calibration of resonance and non-resonance spectra. The pool of compounds that produced positive STD signals were then subdivided into groups of three for retesting. Those compounds that showed STD in both experiments were reordered from Maybridge and tested as single compounds as well as in competitive binding experiments.

To obtain sufficient quantities and stable recombinant full-length BAX for ligand screening, the production process was scaled up to a total culture volume of 48 liters and bacterial pellets were subjected to sequential lysis using a temperature controlled (set at 4 ℃) microfluidizer, followed by binding of the lysate to a chitin affinity resin batch, Dithiothreitol (DTT) elution and purification by size exclusion chromatography. Using this method, 21.6mg BAX protein was produced at a concentration of 0.64mg/mL for the primary screen, representing a total yield of 0.45mg pure full-length protein per liter of bacterial culture. Ligand screening was then performed by Saturation Transfer Difference (STD) NMR to identify molecules that interact with BAX, as described above. STD-NMR measurement of ligands after selective irradiation of target proteins¹H-NMR signal changes, where magnetization transfer from protein to ligand results in a decrease in the signal reflecting ligand-protein interactions.

A Maybridge Ro3 library of 1000 compounds was used for BAX screening. Of the 96 pools analyzed, 37 detected STD positive signals, representing 86 individual search results (hit), were then rescreened in the three pools, resulting in 56 confirmed interactors (fig. 1 b). 53 commercially available compounds were ordered, retested as single items by STD and identified as BAX-interacting fragments (BIF 1-53). The results obtained from STD NMR and liposome release assays are shown in table 1. The structures of the active compounds BIF-1 to BIF-53 are shown in Table 2.

Table 1.

Table 2.

Example 4 Liposome Release assay

Large Unilamellar Vesicles (LUVs) with lipid compositions similar to the outer mitochondrial membrane were formed by liposome extrusion as previously described (see, e.g., Leshchiner et al, Proc. Natl. Acad. Sci. U.S.A.,2013,110: E986-995; Lovell et al, Cell,2008,135: 1074-1084). Briefly, a mixture comprising 48: 28: 10: 10: a lipid mixture of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, dioleoylphosphatidylserine and tetraoleoyl cardiolipin (Avanti Polar Lipids) at a 4 molar ratio. Lipid films were formed by first evaporating the solvent under nitrogen, then by vacuum overnight, followed by storage at-80 ℃ under nitrogen. Lipid membranes were assayed in 1mL of assay buffer (10mM HEPES, 200mM KCl, 1mM MgCl)₂pH 7.0) and mixed with fluorophore and quencher p-8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS, 12.5mM) and p-xylylene bipyridinium bromide (DPX, 45 mM). Liposomes were formed by 5 freeze/thaw cycles, then extruded through a 100nm polycarbonate membrane and purified using a Sepharose CL-2B size exclusion column. To measure BAX activation, BAX (750nM) was added to the indicated concentration of molecular fragments at the indicated time points in the presence of liposomes, followed by the addition of BIM SAHB_A2(750 nM). The assay was performed in black opaque 384-well plates (30. mu.l per well). Release of ANTS/DPX over time was monitored at room temperature using a spectrofluorometer (Tecan Infinite M1000) at an excitation wavelength of 355nm, an emission wavelength of 540nm and a bandwidth of 20 nm. Maximum release was determined by adding Triton X-100 to a final concentration of 0.2% (v/v). Percent release was calculated according to equation 1 below, where F is the observed release and F is₀And F₁₀₀Respectively, baseline and maximum fluorescence.

Formula 1.

((F-F₀)/(F₁₀₀-F₀))×100

To determine whether any of the identified BIFs affected BAX function, 53 BIFs were screened in the above-described liposome release assay designed to identify (1) direct BAX activators and (2) BIM BH3 helices, BIM SAHB_A2(aa 145-164) induced sensitizers or inhibitors of direct BAX activation (see, e.g., Gavatithis et al, Nature,2008,455: 1076-1081). First, baseline fluorescence of liposomes and individual compounds was read, followed by addition of BAX to assess direct activation; then, BIM SAHB_A2Adding to the mixture, and monitoring the effect of the combination, and in the absence of the compound, BIM SAHB_A2And triggering activity of BAX. Using this assay format, 4 direct activators of BAX-mediated liposome release and 8 BIM SAHB were identified_A2Triggered BAX activates sensitizers as shown in table 1. Direct activator Profile with Positive control BIM SAHB_A2Peptides exemplify that the peptides induced time-responsive liposome release in the presence of BAX alone (fig. 1 c). The activity of BIF-44 most significantly reflects the novel sensitizer profile, with minimal effect of BIF-44 on BAX when incubated as a single dose, but with BIM SAHB_A2When used in combination, the maximum BAX-mediated release results from the use of BIM SAHB alone_A2The 50% of (a) is ramped up to 80% of the combined use and shows a faster kinetics (fig. 1 c).

In addition, it was found that BIF-44 sensitization by BAX-mediated liposome release was associated with BIF-44 and BIM SAHB_A2Regardless of the order of addition. Whether in the addition of BIM SAHB_A2While (left) adding BIM SAHB_A2Whether BIMSAHB is added before (to the right)_A2After (intermediate) addition of BIF-44, the same level of BAX activation was achieved, as shown in fig. 11.

Example 5 competitive STD-NMR

The individual compounds were added to 5. mu.M MBAX with or without 5. mu.M of the competing peptide in 20mM potassium phosphate buffer (pH 6.2). STD-NMR was measured as described above. Relative to the absence of peptide, by vmIA or BIM SAHB_A2The competing fragments showed reduced saturation transfer differences in the presence of the peptide.

In prior work to characterize direct BAX activator molecules (i.e., BAM), BAM and BIM SAHB were observed at the BH3 trigger site_A2Direct competition between (see, e.g., Gavatithis et al, nat. chem. biol.2012,8: 639-. In evaluating newly identified BAX sensitizing Activity, it was surprisingly found that BIM SAHB_A2There is no effect on the STD signal (fig. 1d), increasing the possibility of alternative interaction mechanisms for BIF-44.

To assess the structure-based reproducibility and selectivity of observed BIF-44 activity, a series of BIF-44 analogs were evaluated for binding and functional properties. It has been found that BIF-44-like diaryl ethers, which can either substitute the hydroxyl group at the same position with an amine, shift the hydroxyl group to the meta position, or substitute the ether bond with a methylene group, retain BAX binding activity competing with vMIA as evaluated by STD NMR and exhibit strong BAX sensitizing activity (fig. 3a-3 c). In contrast, the diaryl ether with a para-hydroxy group in the second aromatic ring or the diaryl ether with a carboxylate group replacing the hydroxy group of BIF-44 had little or no BAX binding or sensitizing activity (FIGS. 3d-3 e). These data, which provide evidence for structural activity relationships, support the specificity of the role of BIF-44 in binding BAX, competition with vMIA, and sensitization BH 3-mediated activation of BAX.

Example 6 CPMG NMR

CPMG experiments were performed using standard methods (see, e.g., Hajduk et al, J.am.chem.Soc.,1997,119: 12257-12261). NMR analysis BIF-44 was used at a concentration of 300. mu.M with or without the addition of BAX (5. mu.M) in 20mM potassium phosphate buffer, pH 6.2. A 0.5 millisecond delay of tau (tau) is applied (1 millisecond for each CPMG echo period), with the number of echo periods corresponding to 500 milliseconds. As reported, excitation engraving was used for solvent inhibition (see, e.g., Hwang et al, j.magn.reson.a., 1995,112: 275-.

Given the liposome delivery and BIM SAHB_A2Results were obtained for both BIF-44 in the competitive STD secondary screen, and the STD-based BIF-44/BAX interaction results were confirmed using orthogonal NMR measurements (FIGS. 7a-7 b). As described above, the method Carr-Purcell-Meiboom-Gill (CPMG) -NMR, which utilizes the faster T2 relaxation time of proteins compared to ligands, was applied to monitor and comparePotential change in BIF-44 signal after BAX incubation. Formation of the protein-ligand complex reduces the relaxation time of the ligand, resulting in a measurable decrease in the 1H-NMR signal (see, e.g., Dias et al, ACS Med. chem. Lett.2014,5: 23-28; Stockman et al, prog.Nucl. Mag. Reson. Spectrosc.2002,41: 187-. In the presence of BAX, a sharp decrease in signal was observed, indicating BIF-44 binding (fig. 7 c). Furthermore, it has been demonstrated that when using a broad molar ratio of 10 to 175: 1 with BAX, BIF-44 had little or no independent triggering of BAX-mediated liposome release (FIG. 2a), but in BIM SAHB_A2BIF-44 dose reactivity enhanced the kinetics and maximum levels of BAX-mediated liposome release in the presence of this (figure 2 b).

Example 7 Fluorescence Polarization (FP) assay

FITC peptide (25nM) was incubated with serial dilutions of recombinant full-length BAX in binding buffer (20mM potassium phosphate, ph 6.2). For competitive FP, FITC peptide (25nM) was mixed with a fixed concentration of BAX (250nM) and incubated with acetylated peptide or serial dilutions of the compounds described herein. Fluorescence polarization was measured at equilibrium using a SpectraMax M5 microplate reader. Dose response curves were subjected to non-linear regression analysis using Prism software 7 (GraphPad).

Finally, using an alternative method to the competitive fluorescence polarization assay, as originally demonstrated by STD (FIG. 1d), the absence of BIM SAHB was demonstrated for BIF-44 conjugation of BAX_A2Competition. For this experiment, FITC-BIM SAHB_A2And BAX was used as an N-terminal acetylated BIM SAHB_A2Basis for comparison competition with BIF-44 (FIG. 8 a). However for BAX binding, Ac-BIM SAHB_A2With FITC-BIM SAHB_A2Dose-responsive competition was performed, while BIF-44 had little effect (fig. 2 c). Thus, in a series of tertiary screening experiments, it has been determined that BIF-44 binds directly to BAX and does not interact with BIM SAHB_A2Competition, and dose-responsive sensitization of BIM SAHB_A2Triggered BAX-mediated membrane perforation.

vMIA is a cytomegalovirus protein involved in blocking BAX-mediated apoptosis that ensures host cell survival during viral infection and replication (see, e.g., Arnoult et al, proc.natl.acad.sci.u.s.a.2004,101: 7988;. Poncet et al, j.biol.chem.2004,279: 22605-.

Example 8 HSQC NMR

As described previously to produce consistent¹⁵N-labeled recombinant BAX (see, e.g., Suzuki et al, Cell,2000,103: 645-. 25mM sodium phosphate, 50mM NaCl solution, 10% (v/v) D at pH 6.0₂Protein samples with the indicated fragment molar ratios were prepared in O. Correlation of the samples at 25 ℃ on a Bruker 600MHz NMR spectrometer equipped with a cryoprobe¹H-¹⁵N HSQC spectra, processed in Topspin (Bruker) and analyzed using CcpNmr analysis (see, e.g., Vlanken et al, Proteins,2005,59: 687-. The weighted average chemical shift difference is calculated as equation 2, where Δ H/Δ N is for the indicated cross-over peak¹H or¹⁵Change in N (p.p.m).

And (3) formula 2.

Absence of bars indicates no chemical shift difference, or the presence of overlapping or unassigned prolines or residues. BAX cross-peak assignment was applied as previously reported (see, e.g., Suzuki et al, Cell,2000,103: 645-. The significance threshold for chemical shift changes was calculated based on the average chemical shift over all residues plus the standard deviation according to standard Methods (see, e.g., Marintchev et al, Methods Enzymol,2007,430: 283-.

After titration of BIF-44¹⁵N-BAX NMR and identifying a series of focused dose-responsive chemical shift changes co-localized to the very region on BAX associated with the vMIA binding site (FIG. 4a, FIG. 9). the most prominent change (2SD) is located at the junction of the α - α hairpin and the α - α hairpin, juxtaposed to form the binding interface (FIG. 4 b). particularly interesting is the more subtle change (1SD) that becomes enhanced with increasing BIF-44 dose and localized on the internal helical regions of α and 8536 (i.e., the hydrophobic core of BAX) and the adjacent internal interaction surface between α and α (FIGS. 4a-4b and FIG. 9) between the BIF-44 dose, the latter helix being a key BH3 motif that must become everted and exposed to ensure BAX activation and oligomerization.therefore, these NMR data not only demonstrate that the binding of BIF-44 to vMIA at similar interaction sites STD and BIA 6334 complex is further shown by the binding mechanisms of the BIF-44 to the hydrophobic domains of the binding domains of the BIF-19-11-5 motif for the further binding of the inner surface of the complex to form a pseudo-activating and a complex via the binding site of the binding sites of the Biot-19-11-3-8-3-11-5 motif.

Example 9 molecular docking

Calculations show a specific increase in conformational flexibility involving the α 1- α 2 region of BAX, which is far from the BIF-44 docking site, but is affected by allosteric induction, as evidenced by dose-responsive HSQCNMR results (figure 9). figure 15 shows the new binding sites for BAX identified in the measurements described herein.

The Schrodinger software suite (version 2016-2) was used for docking calculations. The conformation of the molecule BIF-44 was generated in a MacroModel using the OPLS3 force field (see, e.g., Harder et al, J.Chem.the facility Compout.2016, 12: 281-. Each of the 20 NMR conformations of Bax (PDB: 1F16) was prepared separately using the default parameters in PrepWiz wizard by Maestro. Docking acceptor grids (radius 1nm) were defined at the center of Ala124 (amino acid with the largest HSQC shift). BIF-44 was then docked on all 20 structures using the Glide Extra Precision (XP) mode (see, e.g., Friesner et al, J.Med.Chem.2006,49: 6177-6196). The gesture that scored the highest was then manually checked for consistency with the experimentally determined HSQC displacement of the complex.

Example 10 molecular dynamics simulation

The first NMR structure of BAX from PDB ID 1F16 was used as the starting structure for the MD calculation. Proteins were prepared using the default parameters of the protein preparation workflow in Maestro (see, e.g., Sastry et al, J.Compout.Aided mol.Des.2013,27: 221-. Protonation states are those states predicted to occur at pH7.0 using the Epik module (see, e.g., Shelley et al, J.Compout.Aided mol.Des.2007,21: 681-. Proteins were pre-soaked in cube boxes of water molecules of TIP3P using The System Builder workflow in Desmond (see, e.g., Jorgensen et al, The Journal of Chemical Physics,1983,79: 926-. The box is sized so that all peptide atoms are at least 1nm from the boundary. All overlapping solvent molecules were removed, the system was neutralized with the appropriate counter ion charge, and 150mM NaCl was then added to simulate buffer conditions. All MD simulations were performed using the Desmond software package and applied OPLS3 force field to model all interactions. The periodic boundary conditions remain constant throughout. Long range electrostatic interactions were calculated using the particle network Ewald method (see, e.g., Esmann et al, J.chem.Phys.1995,103:8577-8593), and van der Waals and short range electrostatic interactions were smoothly truncated at 0.9 nm. A constant system temperature of 300K was maintained using a Nose-Hoover thermostat (see, e.g., Hoover et al, Phys. Rev. A. Gen. Phys.1985,31: 1695-. The motion is integrated using a RESPA integrator (see, e.g., Humphreys et al, J.Phys.chem.,1994,98: 6885-. The default parameters in Desmond are used to relax the system prior to simulation (see, e.g., Guo et al, chem. biol. drug Des.2010,75: 348-. According to this procedure, a production simulation of 100ns was performed and the configuration was saved at 4ps intervals. All simulations were judged to have converged based on the radius of gyration calculation and RMSD.

Example 11 deuterium and hydrogen exchange Mass Spectrometry

Hydrogen-deuterium exchange mass spectrometry (HXMS) experiments were performed as previously described (see, e.g., Barclay et al, mol.cell,2015,57: 873-. Deuterium labeling was added at 18-fold dilution to a pre-equilibrated aliquot (15 min, room temperature) of each BAX protein, molecule, peptide and/or antibody (BAX BH3, Abgent AP1302 a; BAX6A7, Santa Cruz Biotechnology sc-23959) mixture₂O buffer (10mM HEPES, 200mM KCl, 1mM MgCl)₂Pd7.0) for deuterium labeling. At the indicated time points, the samples were quenched by addition of an equal volume of quench buffer (0.8M guanidinium chloride, 0.8% formic acid [ v/v ]]) The labeling reaction was quenched. Each deuterium labeling experiment was repeated at least twice. Proteolysis was performed by incubation with 40 μ g pepsin and 20 μ g factor XIII for 5 min on ice. The digested samples were then processed and analyzed as described previously (see, e.g., Barclay et al, mol. cell,2015,57: 873-. The relative deuterium levels of the identified peptides, which are common to all the conditions evaluated, are shown. The error in average deuterium incorporation for each peptide was determined to be +/-0.25Da or less. The relative deuterium levels of each peptide were calculated by subtracting the average mass of the non-deuterated control samples from the average mass of the deuterium-labeled samples. All mass spectra were processed using DynamX 3.0(Waters Corporation). Deuterium levels are not corrected for reverse exchange and are therefore reported as relative levels (see, e.g., Wales et al, massspectrum. rev.2006,25: 158-.

To assess whether the BIF-44 sensitization mechanism originates from allosteric mobilization of the BAX region α - α (involving BH3 mediated initiation of BAX activation through the N-terminal trigger site), a comparative hydrogen-deuterium exchange mass (HXMS) spectrum as described above was performed on a mixture of BAX and liposomes in the presence or absence of BIF-44 HXMS probes the protein structure by measuring deuterium incorporation of the backbone amide hydrogen (see, e.g., Engen et al, anal. chem.2009,81: 7870-7875). when diluted in deuterium buffer, the backbone hydrogen of the flexible and/or exposed protein region would rapidly exchange with deuterium, whereas the buried domain and/or the region containing hydrogen bonds involving the backbone amide hydrogen (e.g., in the α helix) shows a slowed or inhibited deuterium exchange (see, e.g., Laiken et al, Biochemistry,1969,8: 519; Prikz et al, Protic. Acnl. 85l. 8: 52. cndot. as a specific inhibition of the BH 52-mediated binding to the BH 52 region of the BH 52-19 region; the motif-19, the ligand binding to the polypeptide region of the polypeptide fragment of the polypeptide molecule can be verified by the inhibition of the biochemical pathway of the BH 19, the biochemical pathway of the polypeptide binding to the polypeptide binding of the polypeptide-19, where the polypeptide binding of the polypeptide binding to the polypeptide, such a-19, the polypeptide, such as opposed to the polypeptide, such a domain, such as observed in the polypeptide, where the inhibition of the biochemical pathway of the polypeptide, the biochemical pathway, the pathway of the biochemical pathway, the pathway of the pathway, the pathway of the pathway, polypeptide, the pathway of the pathway, polypeptide, the pathway of.

To study BIF-44 and BIM SAHB_A2How conjugation at different sites acts synergistically to trigger BAX activation, at BIF-44, BIM SAHB_A2Or a combination thereof, performing HXMS analysis on the BAX. BIM SAHB_A2And BIF-44, which are consistent with their different binding sites and different mechanisms of action. And BIM SAHB_A2Directly merging with α -helix 1 and α -helix 6Binding of the N-terminal trigger site and substitution of the α 1- α 2 loop results in 6A7 epitope exposure (see, e.g., Gavatithis et al, mol. cell,2010,40: 481-492; Barclay et al, mol. cell,2015,57: 873-886). BIF-44 engages the distal site, causing local allosteric changes in the loops at the distal α 1- α 2 and BH3 α 2 helices (FIG. 6 a). Combined treatment significantly enhances deprotection of substantially the entire α 1- α 2 region (FIGS. 6a-6b), which in combination with BIF-44 effectively sensitizes BIM SAHB_A2The ability to mediate conformational activation of BAX is consistent.

Example 12 mitochondrial cytochrome c Release assay

Separation from Alb^CreBax^f/fBak^-/-Liver mitochondria (0.5mg/mL) in mice and release assays were performed as described (see, e.g., Walensky et al, mol. cell,2006,24: 199-. Briefly, mitochondria were incubated with 100nMBAX, 250nM BIM SAHB_A2And/or BIF-44 at the indicated concentration in assay buffer (200mM mannitol, 68mM sucrose, 10mM HEPES-KOH [ pH7.4 ]) at room temperature]110mM KCl, 1mM EDTA, protease inhibitor) for 45 minutes (see, e.g., Llambi et al, mol. cell,2011,44: 517-531). The pellet and supernatant fractions were separated by centrifugation and a colorimetric ELISA (R) was used&D Systems) to quantify cytochrome c. The percentage of cytochrome c released into the supernatant (% cytochrome c release) was calculated according to formula 3, wherein cytochrome c is released_supAnd cytochrome c_maxRepresents the amount of cytochrome c detected in the supernatant of a compound-treated sample or a 1% (v/v) Triton X-100-treated sample, respectively.

And (3) formula.

% cytochrome c Release ═ cytochrome c_sup]/[ cytochrome c_max]*100

Finally, to link these interesting mechanism findings to the physiological environment, the ability of BIF-44 to sensitize BAX-mediated mitochondrial apoptosis was tested as measured by cytochrome c release from liver mitochondria in treated mice. BIF-44 dose-reactively sensitized BIM SAHB consistent with a synergistic effect in BAX N-terminal region conformational activation as observed by HXMS_A2Induction of trigger BAX-mediated cytochrome c Release from mitochondria(FIG. 6 c). Thus, NMR screening identified small molecule BAX sensitizers that promoted the initiation of BH 3-mediated direct BAX activation through a novel allosteric mechanism.

EXAMPLE 13 Isothermal Titration Calorimetry (ITC)

Binding Affinity was measured by adding 0.15mM recombinant BAX protein to the cells and injecting 2.0 μ L of 1.0mM ligand by syringe for a total of 30 injections at 25 ℃ using Affinity ITC (TA instrument). BAX and BIF-44 solutions were prepared in 20mM potassium phosphate buffer (pH6.2) at a final concentration of 2% (v/v) DMSO. Prior to titration, the samples were centrifuged for 15 minutes at 4 ℃. The ITC experiment was repeated twice and the data were analyzed using the single binding site model using the NanoAnalyze software package (TA instruments) and thermodynamic parameters were calculated according to equation 4, where Δ G, Δ H and Δ S are changes in free energy, enthalpy and entropy of binding. The ITC measurement results are shown in fig. 10.

And (4) formula 4.

ΔG＝ΔH-TΔS＝-RTlnK_B

Example 14 NMR-based detection of Small molecule aggregates

For detecting line broadening, obtaining standards¹H-NMR spectrum. The T2 decay curve was generated by measuring the CPMG NMR spectrum of the molecule as performed above. The number of echo cycles corresponds to the decay time. The intensity of the fragrance peak was measured at the specified decay time and normalized to a maximum intensity of 1at 10ms decay time. Curves were fitted to the monophasic decay model using Prism software (Graphpad). The excitation engraving is used for solvent inhibition. Samples for both assays were in 20mM potassium phosphate buffer, pH6.2, 10% (v/v) D₂Is prepared in O. The results of these measurements are depicted in fig. 12-13.

Example 15 dynamic light Scattering

Samples were measured at room temperature on a DynaPro-99 instrument at a 90 ° probe angle with a 10 second acquisition time for each measurement. Compounds were diluted from 100mM stock (pH6.2 in 20mM potassium phosphate buffer, final DMSO concentration of 1%). The results of the dynamic light scattering measurements are shown in fig. 14.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. It will be appreciated by persons of ordinary skill in the art to which the invention relates that any feature described herein with respect to any particular embodiment of the invention may be combined with one or more features of any other feature of any other embodiment of the application described herein, with appropriate modification to ensure compatibility of the combination. Such combinations are considered part of the present application.

Sequence listing

<110> Dana Faber cancer institute

<120> small molecule sensitization to BAX activation for inducing cell death

<130>00530-0334P01

<140>62/471,174

<141>2017-03-14

<160>3

<170>PatentIn version 3.5

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<213>Homo sapiens

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Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser

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Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys

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Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala

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Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly

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<223> description of artificial sequences: synthetic peptides

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20

Claims

1. A composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof

And one or more pharmaceutically acceptable carriers, wherein:

L¹selected from the group consisting of bond, C_1-3Alkylene, -O-, -O (C)_1-3Alkylene) -, C_1-3Cyanoalkylene, -S-, -SO₂-、-S(C_1-3Alkylene) -and-c (o) -or a pharmaceutically acceptable salt thereof;

R¹selected from halogen, OH, C_1-3Alkyl radical, C_1-3Haloalkyl, NH₂CN, phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkylWherein said phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl are each optionally substituted with 1,2 or 3 independently selected R^ASubstituted by groups;

R²selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups;

R³selected from H, halogen, OH, NH₂、C(O)C_1-3Alkyl and C (S) C_1-3Alkyl groups;

R⁴selected from H, halogen, OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Haloalkyl and O (C)_1-3Cyanoalkyl);

R⁵selected from H, halogen, OH, NH₂And C (O) C_1-3Alkyl groups;

R⁶selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups;

each R^AIndependently selected from OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Hydroxyalkyl, C (O) OH, C (O) C_1-3Alkyl and C (O) N (C)_1-3Alkyl radical)₂Wherein said C_1-3Alkyl is optionally substituted by NH₂And (4) substitution.

2. The composition of claim 1, wherein L¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-C (O) -in a pharmaceutically acceptable carrier.

3. The composition of claim 1, wherein L¹is-O-, -CH₂-or-OCH₂-。

4. The composition of any one of claims 1 to 3, wherein R¹Selected from the group consisting of Cl, CH₃、CF₃、NH₂CN, phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, wherein said phenyl, 5-6 membered heteroaryl and 5-6 membered heterocycle areEach alkyl group optionally substituted with 1 or 2 independently selected R^AAnd (4) substituting the group.

5. The composition of any one of claims 1 to 3, wherein R¹Selected from the group consisting of Cl, CH₃、CF₃、NH₂CN, phenyl, pyridyl, furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, pyrazolyl, 1,2, 4-thiadiazolyl, piperidinyl, morpholinyl, and 4, 5-dihydrothiazolyl, wherein said phenyl, pyridyl, furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, pyrazolyl, 1,2, 4-thiadiazolyl, piperidinyl, morpholinyl, and 4, 5-dihydrothiazolyl are each optionally substituted with 1 or 2 independently selected R^AAnd (4) substituting the group.

6. The composition of any one of claims 1 to 5, wherein each R is^AIndependently selected from OH, NH₂、CN、CH₃、CH₂OH、CH₂CH₂NH₂、C(O)OH、C(O)CH₃And C (O) N (CH)₃)₂Group (d) of (a).

7. The composition of any one of claims 1 to 3, wherein R¹Is phenyl, optionally substituted by 1 or 2 independently selected R^AAnd (4) substituting the group.

8. The composition of any one of claims 1 to 3, wherein R¹Is phenyl, 4-hydroxyphenyl, 3-hydroxyphenyl, 4-aminophenyl, 4-carboxyphenyl or 4-hydroxymethylphenyl.

9. The composition of any one of claims 1 to 8, wherein R²Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

10. The composition of any one of claims 1 to 8, wherein R²Is H or CH₃。

11. The composition of any one of claims 1 to 10, wherein R³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃Group (d) of (a).

12. The composition of any one of claims 1 to 10, wherein R³Is H.

13. The composition of any one of claims 1 to 12, wherein R⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN.

14. The composition of any one of claims 1 to 12, wherein R⁴Is H or OH.

15. The composition of any one of claims 1 to 14, wherein R⁵Selected from H, F, Cl, NH₂And C (O) CH₃Group (d) of (a).

16. The composition of any one of claims 1 to 14, wherein R⁵Is H or NH₂。

17. The composition of any one of claims 1 to 16, wherein R⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

18. The composition of any one of claims 1 to 16, wherein R⁶Is H.

19. The composition of claim 1, wherein the compound of formula I is selected from the group consisting of:

20. the composition of claim 1, wherein the compound of formula I is:

or a pharmaceutically acceptable salt thereof.

21. A composition comprising a compound of formula II or a pharmaceutically acceptable salt thereof

And one or more pharmaceutically acceptable carriers, wherein:

X¹is NH or S;

X²is C or N;

L¹selected from the group consisting of a bond, -C (O) -, -C (O) O-and-SO₂-a group of compositions;

R¹selected from the group consisting of C_1-3Alkyl, NH₂Two (C)_1-3Alkyl) amino and 5-6 membered heterocycloalkyl;

R²selected from H, halogen, C_1-3Alkyl and C (O) OC_1-3Alkyl groups;

R³selected from the group consisting of H, C_1-3Alkyl and 5-6 membered heteroaryl; or when X is²When is N, R³Is absent; and

R⁴selected from H and C_1-3Alkyl groups.

22. The composition of claim 21, wherein X¹Is NH.

23. The composition of claim 21, wherein X¹Is S.

24. The composition of any one of claims 21 to 23, wherein X²Is C.

25. The composition of any one of claims 21 to 23, wherein X²Is N.

26. The composition of any one of claims 21-25, wherein R¹Is selected from the group consisting of CH₃、CH₂CH₃、NH₂、N(CH₂CH₃)₂Piperidinyl and dihydrothiophen-3 (2H) -onyl.

27. The composition of any one of claims 21 to 25, wherein-L¹-R¹Forming a group selected from the group consisting of: NH (NH)₂、C(O)OCH₃、C(O)OCH₂CH₃、C(O)N(CH₂CH₃)₂、SO₂-piperidinyl and dihydrothiophen-3 (2H) -onyl.

28. The composition of any one of claims 21-27, wherein R²Selected from H, Cl, CH₃And C (O) OCH₃Group (d) of (a).

29. The composition of any one of claims 21-28, wherein R³Selected from the group consisting of H, CH₃、CH₂CH₃And thienyl.

30. The composition of any one of claims 21-29, wherein R⁴Selected from H and C_1-3Alkyl groups.

31. The composition of claim 21, wherein the compound of formula II is selected from the group consisting of:

32. a composition comprising a compound of formula III or a pharmaceutically acceptable salt thereof

And one or more pharmaceutically acceptable carriers, wherein:

a single bond or a double bond;

ring A is selected from the group consisting of 5-6 membered cycloalkyl, 5-6 membered heteroaryl and 5-6 membered heterocycloalkyl, forming a fused ring with ring B, wherein ring A is optionally substituted with 1,2 or 3 independently selected R^ASubstituted by groups;

R¹selected from the group consisting of H, C (O) OC_1-3Alkyl, OC (O) C_1-3Alkyl, C (S) NH₂And ═ N-OH;

R^1ais H; or when R is^1aWhen the carbon atom to which it is attached forms a double bond, R^1aIs absent;

R²selected from the group consisting of H and halogen;

R^2ais H; or when R is^2aWhen the carbon atom to which it is attached forms a double bond, R^2aIs absent;

R³selected from H, halogen, C_1-3Alkyl radical, C_1-3Hydroxyalkyl, NHC (O) C_1-3Alkyl and (C)_1-3Alkylene) NHC_1-3Alkyl groups;

R^3ais C_1-3An alkyl group; or when R is^3aWhen the carbon atom to which it is attached forms a double bond, R^3aIs absent;

R⁴selected from H and C_1-3Alkyl groups;

R^4ais H; or when R is^4aWhen the carbon atom to which it is attached forms a double bond, R^4aIs absent; and

each R^AIndependently selected from the group consisting of ═ O, ═ S, CN and C_1-3Alkyl radical, C_1-3Hydroxyalkyl radical, S (C)_1-3Alkyl) and C (O) OH.

33. The compound of claim 32, wherein ring a is 5-6 membered heteroaryl, optionally substituted with 1,2, or 3 independently selected R^AAnd (4) substituting the group.

34. The compound of claim 32, wherein ring a is 5-6 membered heterocycloalkyl, optionally substituted with 1,2, or 3 independently selected R^AAnd (4) substituting the group.

35. The compound of any one of claims 32 to 34, wherein each R^AIndependently selected from the group consisting of ═ O, ═ S, CN, CH₃、CH₂OH、SCH₃And C (O) OH.

36. The compound of claim 32, wherein ring a is unsubstituted 5-6 membered cycloalkyl.

37. The compound of claim 32, wherein ring a is selected from the group consisting of:

wherein each one

Both represent a bond linking fused ring a and ring B.

38. The method of any one of claims 32 to 37The composition of (1), wherein R¹Selected from the group consisting of H, C (O) OCH₃、OC(O)CH₃、C(S)NH₂And N-OH.

39. The composition of any one of claims 32-38, wherein R²Selected from the group consisting of H and Cl.

40. The composition of any one of claims 32-39, wherein R^2aIs H.

41. The composition of any one of claims 32-39, wherein R^2aIs absent.

42. The composition of any one of claims 32-41, wherein R³Selected from H, Cl, CH₃、CH₂OH、NHC(O)CH₃And CH₂NHCH₃Group (d) of (a).

43. The composition of any one of claims 32-42, wherein R^3aIs CH₃。

44. The composition of any one of claims 32-42, wherein R^3aIs absent.

45. The composition of any one of claims 32-44, wherein R⁴Selected from the group consisting of H and CH₃Group (d) of (a).

46. The composition of claim 32, wherein the compound of formula III is selected from the group consisting of:

47. a composition comprising a compound comprising a moiety of formula IV:

and one or more pharmaceutically acceptable carriers, wherein:

R¹selected from the group consisting of phenylene, 5-6 membered heteroarylene, and 5-6 membered heterocycloalkylene, each of which is optionally substituted with 1,2, or 3 independently selected R^ASubstituted by groups;

R²selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups;

R⁵selected from H, halogen, OH, NH₂And C (O) C_1-3Alkyl groups;

R⁶selected from H, halogen, OH, CN, C_1-3Alkyl and C (O) OC_1-3Alkyl groups;

each R^AIndependently selected from OH, NH₂、CN、C_1-3Alkyl radical, C_1-3Hydroxyalkyl, C (O) OH, C (O) C_1-3Alkyl and C (O) N (C)_1-3Alkyl radical)₂Wherein said C_1-3Alkyl is optionally substituted by NH₂Substitution。

48. The composition of claim 47, wherein L¹Is selected from the group consisting of a bond, -CH₂-、-O-、-OCH₂-、-CH(CN)-、-S-、-SO₂-、-SCH₂-and-C (O) -in a pharmaceutically acceptable carrier.

49. The composition of claim 47 or 48, wherein R¹Is phenylene optionally substituted with 1 or 2 independently selected R^AAnd (4) substituting the group.

50. The composition of any one of claims 47-49, where each R^AIndependently selected from OH, NH₂、CN、CH₃、CH₂OH、CH₂CH₂NH₂、C(O)OH、C(O)CH₃And C (O) N (CH)₃)₂Group (d) of (a).

51. The composition of any one of claims 47-50, wherein R²Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

52. The composition of any one of claims 47-51, wherein R³Selected from H, F, Cl, NH₂、C(O)CH₃And C (S) CH₃Group (d) of (a).

53. The composition of any one of claims 47-52, wherein R⁴Selected from H, Cl, NH₂、CN、CH₃、CF₃And OCH₃CN.

54. The composition of any one of claims 47-53, wherein R⁵Selected from H, F, Cl, NH₂And C (O) CH₃Group (d) of (a).

55. The combination of any one of claims 47 to 54Wherein R is⁶Selected from H, Cl, CN, CH₃And C (O) OCH₃Group (d) of (a).

56. A method of sensitizing and/or activating the pro-apoptotic activity of BAX comprising contacting a cell sample or tissue sample comprising BAX with the composition of any one of claims 1 to 55.

57. A method of sensitizing and/or activating the pro-apoptotic activity of BAX in a subject comprising administering to the subject a composition of any one of claims 1 to 55.

58. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1 to 55.

59. The method of claim 58, wherein the cancer is selected from the group consisting of: breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck malignancy, breast malignancy, ovarian malignancy, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder malignancy, pancreatic malignancy, gastric cancer, colon malignancy, prostate malignancy, genitourinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical cancer, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, neuroblastoma, rhabdomyosarcoma, sarcoid sarcoma, and malignant melanoma, Kaposi's sarcoma, polycythemia vera, primary thrombocythemia, hodgkin's disease, non-hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

60. The method of claim 58, wherein the cancer is leukemia.

61. The method of claim 60, wherein the leukemia is selected from the group consisting of: acute lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

62. The method of claim 60, wherein the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphoblastic leukemia, and chronic myelogenous leukemia.

63. A method of identifying a compound that sensitizes and/or activates the pro-apoptotic activity of a BAX polypeptide, the method comprising:

64. The method of claim 63, wherein the determining step is performed by saturation transfer difference NMR, HSQC NMR, surface plasmon resonance, biolayer interferometry, or competitive fluorescence polarization assay.

65. The method of claim 63 or 64, wherein binding of the compound to the BAX polypeptide results in a change in signal from the NMR spectrum of the compound.

66. The method of any one of claims 63-65, further comprising detecting activation of the BAX polypeptide by the compound.

67. The method of claim 66, wherein the detecting step comprises performing an assay selected from the group consisting of: detecting BAX oligomerization, antibody-based BAX conformation detection, mitochondrial cytochrome c release assay, liposome release assay, cell death assay, mitochondrial or cell morphology assay, mitochondrial calcium flux assay, mitochondrial transmembrane quantification assay, and quantification of caspase 3 activity or annexin V binding.

68. The method of any one of claims 63-67, wherein the compound binds to the binding site with an affinity of <1 mM.

69. The method of any one of claims 56 to 68, wherein said compound sensitizes activation of pro-apoptotic activity of said BAX polypeptide.

70. The method of any one of claims 56-68, wherein the compound activates a pro-apoptotic activity of the BAX polypeptide.

71. The method of any one of claims 56-70, wherein said method further comprises administering an additional therapeutic agent that activates BAX pro-apoptotic activity.

72. The method of claim 71, wherein the additional therapeutic agent is BIM SAHB_A2。